Remedy for bovine leukemia prophylaxis and use thereof

ABSTRACT

The invention generally relates to the field of veterinary medicine and can be used for bovine leukemia prevention. The invention expands the range of means of the claimed application and provides lifelong resistance of animals to the leukemia virus. 
     The invention is aimed at solving the problem of expanding the range of preventive medications and forming new approaches to the problem of preventing bovine leukemia, which allow ensuring lifelong resistance of the animals to leukemia infection. 
     The technical result consists in the design of a remedy and use thereof for bovine leukemia prevention, which would allow providing a targeted immune response, efficiently and with minimal costs, by activating cellular immunity against bovine leukemia virus by increasing the number of killer cells. 
     The remedy for bovine leukemia prevention contains a water-soluble protein fraction with a molecular weight of 18-20 kDa, isolated from the tuberculosis mycobacterium destruction products, characterized by the presence of peaks at a wavelength of 214 nm in the UV region, a phosphate-buffered saline, an aqueous formaldehyde solution, and an isotonic sodium chloride solution. 
     The use consists in administering the said remedy to 1 to 9 days old calves with a single intramuscular injection at a dose of 4.0-6.0 ml per capita.

CROSS-REFERENCE TO RELATED APPLICATIONS

The instant application is a National Phase Entry of InternationalPatent Application No. PCT/RU2017/000404, filed on Jun. 13, 2017, andclaims priority to Russian Patent Application No. 2016145862, filed onNov. 22, 2016, the entire specifications of both of which are expresslyincorporated herein by reference.

FIELD OF THE INVENTION

The invention generally relates to the field of veterinary medicine andcan be used to prevent bovine leukemia. The disclosure expands the rangeof means of the claimed application and provides the lifelong resistanceof animals to bovine leukemia virus (BLV).

BACKGROUND OF THE INVENTION

Several remedies are hitherto known to prevent bovine leukemia and thereare methods for preventing this disease using these medications.

An inactivated vaccine against bovine leukemia is known (see USSRauthor's certificate No 820,015, class A61K39/12, publ. Oct. 23, 1987)containing the protein fractions p25 and gP70 isolated from theperipheral blood leukemia cells of animals suffering from leukemia.

The obtained antigen-protein immunizing agent with Freund's completeadjuvant in a 1:1 ratio is administered to 1 to 3 months old calves.This immunization is carried out three times at intervals of 10-12 days.

Another inactivated vaccine against bovine leukemia is also disclosed(see RU patent No 2,202,367, IPC class A61K39/12, publ. Apr. 20, 2003),containing virus-containing leukocyte cells of the peripheral blood ofanimals. The immunization of animals is carried out subcutaneously threetimes with an interval of 10-14 days. Animals are vaccinated at the ageof 2 months to 2 years.

These vaccines, however, have not found wide practical application dueto the lack of means of immunological failure correction and theformation of low-titer antibodies.

Various remedies for bovine leukemia prevention are known as well.

In particular, the use of an amber biostimulator (see RU patent No2,420,275, IPC class A61K31/194, publ. Jun. 10, 2011),polycarboxyethylene (see RU patent No 2,421,184, IPC class 2421184,publ. Jun. 20, 2011), metronidazole and norsulfazole or sulphadimezine(see RU patent No 2,300,883, IPC class A61K67/02, publ. Jun. 20, 2007),ligfol (see RU patent No 2,320,357, IPC class A61K36/00, publ. Mar. 27,2008) etc. is known.

However, the influence of these preparations on the immune system atleukemia has not been studied and these remedies have found no practicalapplication.

More particularly, metronidazole and sulfonamides have bactericidalrather than virucidal action, which does not allow them to render aspecific effect on BLV. Ligfol is a hydrolyzate of natural lignin, humicsubstances being its active ingredient. Ligfol shows stress-correctorand adaptogenic actions, its injections are painful and cause long-termanxiety of animals. The effect of the amber biostimulator, which is amixture of succinic acid and Dorogov's antiseptic stimulant (DAS),fraction No. 2, like that of any biologically active drug, has also beenstudied little and requires additional studies.

The closest to the present invention is the bovine leukemia preventionon the basis of tuberculosis toxoid (see RU patent No 2,396,978, IPCclass A61K39/295, publ. Aug. 20, 2010). Tuberculosis toxoid isadministered subcutaneously 20-30 days prior to vaccination with aninactivated vaccine against bovine leukemia. This tuberculosis toxoid isobtained (see, e.g., RU Patent No. 2,392,002, IPC class A61K39/00, publ.Jun. 20, 2010) by detoxification of tubercular exotoxins and endotoxinswith two detoxifiers, namely, 0.2% formalin solution during 7-9 days at42-45° C. and 0.5% aethonium solution during 7-9 days at 42-45° C.

However, this method for preventing bovine leukemia uses themycobacteria destruction products in the whole, rather than the proteinfraction isolated therefrom. The degradation product generally containsproteins, lipids, and polysaccharides. Lipids and polysaccharidesincrease the immunogenic load on the animal organism, change thehomeostasis state and prevent a targeted immune response to mycobacteriaproteins, which, in the end, reduces the efficiency of such preventivemeasures and does not ensure lifelong resistance of animals to thedisease.

SUMMARY OF THE INVENTION

The invention is aimed at solving the problem of expanding the range ofpreventive means and forming new approaches to the problem of preventingbovine leukemia, which allow ensuring lifelong resistance of animals toleukemia infection.

The technical result achieved is the design of a remedy and use thereoffor bovine leukemia prevention, which would allow providing a targetedimmune response, with efficiency and minimal costs, by activatingcellular immunity against bovine leukemia virus by increasing the numberof killer cells.

To solve the task posed and to achieve this technical result, a group ofclaims is proposed, namely, a remedy for bovine leukemia prevention anda use thereof.

The technical result as to the remedy itself is achieved in that theremedy for preventing bovine leukemia, according to the disclosure,contains a water-soluble protein fraction with a molecular weight of18-20 kDa isolated from the tuberculosis mycobacterium destructionproducts and characterized by the presence of peaks at a wavelength of214 nm in the UV region, a phosphate-buffered saline, an aqueousformaldehyde solution, and an isotonic sodium chloride solution with thefollowing component concentrations, wt %:

the water-soluble protein fraction with the molecular weight of 18-20kD, isolated from the tuberculosis mycobacterium destruction products0.05-0.125,

the phosphate-buffered saline 9.95-24.875 the aqueous formaldehydesolution 0.025-0.046, and the isotonic sodium chloride solution therest.

The said technical result, as to the use of this remedy, is achieved inthat 1 to 9 day old calves are single injected intramuscularly, at adose of 4.0-6.0 ml, the preparation containing the water-soluble proteinfraction with the molecular weight of 18-20 kDa isolated from thetuberculosis mycobacterium destruction products and characterized by thepresence of peaks at the wavelength of 214 nm in the UV region, thephosphate-buffered saline, the aqueous formaldehyde solution, and theisotonic sodium chloride solution with the following concentrations ofthese components, wt %: the water-soluble protein fraction with themolecular weight of 18-20 kD, isolated from the tuberculosismycobacterium destruction products 0.05-0.125,

the phosphate-buffered saline 9.95-24.875, the aqueous formaldehydesolution 0.025-0.046, and the isotonic sodium chloride solution therest.

The indicated features are significant and sufficient to obtain theclaimed technical result.

The known sources of patent and scientific-technical informationdescribe no preparations to prevent bovine leukemia, which would ensurelifelong resistance of animals to leukemia infection by activating theircellular immunity by increasing the number of killer cells. The mixtureof the water-soluble protein fraction with the molecular weight of 18-20kDa isolated from the tuberculosis mycobacterium destruction products inthe phosphate-buffered saline with solutions of formaldehyde and sodiumchloride provides activation of the killer defense system, namely,involvement of the T-lymphocyte (Th₀) subpopulation into immuneresponse.

In the claimed remedy for bovine leukemia prevention, the mixture of aformaldehyde solution with an isotonic sodium chloride solution is usedas a component providing stabilization of immune response at the killersystem level rather than as a detoxifier.

Introduction of the only protein fraction or only formaldehyde does notgive the desired result. Only the synergism of the components and theirexperimentally chosen concentrations make it possible to obtain a higheffect of animals' resistance to leukemia infection by activating theirkiller system. Moreover, the formaldehyde concentration does not changethe structure of the protein component of mycobacteria.

We have found a new, unobvious approach to solving the problem ofpreventing bovine leukemia by using a mixture of components (the proteinfraction with a certain molecular weight extracted from the tuberculosismycobacterium destruction products and a mixture of solutions offormaldehyde and sodium chloride) to increase the number of killer cellsof the immune system.

Killer cells are known to inhibit the development of tumor cells (see,for example, RU patent No 2,443,411, IPC class A61K9/08, publ. Feb. 27,2012, RU patent No 2,262,941, IPC class A61K35/16, publ. Oct. 27, 2005).However, they are used exclusively to treat the disease.

The use of the mechanism of increasing the number of killer cells of theimmune system to prevent leukemia is hitherto unknown and has not beendisclosed anywhere.

The use of any protein fraction isolated from the destruction productsof tuberculosis mycobacterium to prevent leukemia is also unknown.

Administration of our remedy to calves up to 10 days of age (i.e., inthe neonatal period) on the basis of a mixture of an isolated proteinfraction with a formaldehyde solution for bovine leukemia prevention isalso unknown.

Treatment of calves in their neonatal period by this remedy is explainedby the fact that the humoral immunity is absent in such calves at birth,while the cellular one is already sufficiently developed. Cellularimmunity activation at such an early age and, as a consequence,activation of the killer system of immunity, provides lifelongresistance of animals to leukemia infection.

Cytotoxic T-lymphocytes CD8 and inflammatory T-lymphocytes CD4 (Th1) areknown to be responsible for cellular immunity, whilst T-helperlymphocytes CD4 (Th2) and B-lymphocytes are responsible for humoralimmunity, respectively.

The present invention solves the problem of activating effectiveimmunity against bovine leukemia by increasing cytotoxic lymphocytes(CD8) and inflammatory T-lymphocytes CD4 (Th1), decreasing T-helperlymphocytes CD4 (Th2), and increasing the number of the thirdsubpopulation of Th lymphocytes (Th0).

Therefore, proper conditions in the organism of animals are created inadvance for increasing the number of killer cells, which, being abarrier, inhibit the development of tumor cells. This mechanism allowsthe balance between cellular and humoral immunity to be preserved, whichwill be maintained throughout life.

The foregoing allows us to conclude on an inventive step criterion inthe claimed invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure is illustrated in FIG. 1, which shows the chromatogram ofthe protein fraction isolated from the destruction products of thecausative agent of tuberculosis in cattle M. bovis (strain No. 8).

DETAILED DESCRIPTION

The remedy to prevent bovine leukemia is prepared as follows. As thedestruction products of the causative agent of tuberculosis, PPDtuberculin for mammals is used, obtained from the strain M. bovis No. 8and representing a clear liquid of light brown color.

To obtain the protein fraction, the liquid with the destruction productsof the pathogen is precipitated with a saline solution (for example,ammonium sulfate) and centrifuged. The supernatant is then removed andthe precipitate is redissolved in a saline buffer solution.

Dialysis is carried out, and the pH of the solution is adjusted to7.0-7.4, after which the protein concentration is analyses byconventional methods, in particular, by the Lowry method.

The choice of such pH values of the solvent is explained by thecorresponding value in the body cells. It is well known that pH in thebody cells and the extracellular fluid is about 7.0 and 7.4,respectively.

The molecular weight of the protein was determined by high pressureliquid chromatography (HPLC), which was carried out, in particular, on aStayer liquid chromatograph, using a spectrophotometric detector A214(the wavelength 214 nm). A BioSep-SEC-S 2000 column (5 μm 145 A, 300×7.8mm) was used for separation. 0.01 M phosphate-buffered saline was usedas the eluent, the flow rate was 1 ml/min, or 0.01 M phosphate-bufferedsaline with 0.025% sodium azide solution (pH 6.8) and at a flow rate of2 ml/min.

Bovine serum albumin with a molecular weight within 64-70 kDa,lactoferrin with a molecular weight of 75-80 kDa, and papain with amolecular weight of 20 kDa were used as standards.

Standard values were taken into account, such as the retention time ofthe sample on the column, or the retraction time (RT, min), the peakintensity by height (mAU), and the relative quantitative content of thesubstance, which was calculated as the percentage ratio of each peak tothe total area of the peaks (A, %).

The major proteins in the fraction isolated from the destructionproducts of the causative agent of bovine tuberculosis M. bovis (strainNo. 8) had molecular weights within 18-20 kDa (see FIG. 1).

Table 1 shows the result of chromatography of the composition based onthe protein fraction isolated from the destruction products of thecausative agent of bovine tuberculosis.

TABLE 1 Mol. Time, Height, Area, Resolution, n, weight, No. min mAU mAU· s n + 1 A, % kDa 1. 18.54 100.18 3234.48 0.00 100 18-20

Next, the resulting protein fraction with the molecular weight of 18-20kDa is mixed, in a buffer solution containing 4.8-5.2 mg/ml of theprotein (i.e., 0.48-0.52%), with an isotonic sodium chloride solution,of a concentration within 0.85-0.95%.

To prepare a 50% solution of the protein fraction, 1 part of the buffersolution with the protein is taken and 1 part of the isotonic solutionis added.

The composition of the resulting mixture is as follows:

the protein fraction—0.24-0.26%,

the phosphate-buffered saline—49.74-49.76%, and

the sodium chloride solution—the rest.

When preparing a 20% solution of the protein fraction, the compositionof the resulting mixture will be as follows:

the protein fraction—0.096-0.104%,

the phosphate buffered saline—49.896-49.904%, and

the sodium chloride solution—the rest.

Next, a mixture of an aqueous formaldehyde solution with an isotonicsodium chloride solution is prepared.

To do this, 0.15-0.25 ml of a 36.5-37.5% medical solution of formicaldehyde (formaldehyde) is taken and added to 99.75-99.85 ml of anisotonic 0.85% (or 0.95%) solution of sodium chloride. The mixture ofthese solutions is stirred thoroughly.

The ratio of the components of the formaldehyde and isotonic sodiumchloride solutions was selected experimentally.

When preparing the remedy from 37% formaldehyde solution, the finalformaldehyde concentration in the resulting composition will be0.055-0.099 wt %.

Then the resulting mixture of the solutions of the protein fraction inthe buffer and isotonic solutions is mixed with the mixture of theformaldehyde and isotonic sodium chloride solutions.

Example 1. Preparation of the Remedy for Leukemia Prophylaxis

100 ml of PPD tuberculin for mammals, obtained from M. bovis strain No.8 is taken. The protein fraction is prepared therefrom as follows.

42 g of ammonium sulfate is added to 100 ml of tuberculin to precipitatethe protein, centrifuged at 4,500 rpm during 20 min. The supernatant isremoved and the precipitate is redissolved in 1 ml of a 0.01 Mphosphate-buffered saline. The system is dialyzed by means of a dialysismembrane with a pore diameter of 3,500 Da in a 0.01 M phosphate-bufferedsaline during 24 h, the pH of the solution is adjusted to 7.2-7.4, afterwhich the protein concentration is determined by the Lowry method usinga StatFax 3300 biochemical analyzer.

The concentration of the protein fraction from the destruction productsof the causative agent of bovine tuberculosis was 5.0 mg/ml.

Analogously to Example 1, protein fractions from other batches of PPDtuberculin were obtained. The amount of protein everywhere varied from4.8 to 5.2 mg/ml.

Next, the resulting protein fraction with the molecular weight of 18-20kDa in the buffer solution containing 5 mg/ml protein (i.e. 0.5%) ismixed with the isotonic sodium chloride solution (0.85-0.95%).

A mixture of the aqueous formaldehyde solution and the isotonic sodiumchloride solution is then prepared.

To do this, 0.2 ml of a 37% medical solution of formic aldehyde(formaldehyde) is taken and added to 99.8 ml of an isotonic (0.85%)sodium chloride solution. The mixture of these solutions is thoroughlystirred. The final formaldehyde concentration in the resultingcomposition will be 0.074 wt %.

The protein fraction in the phosphate-buffered saline is mixed with themixture of solutions of formaldehyde and sodium chloride in equalproportions (1:1).

The contents of the components in the composition will be as follows, wt%:

the water-soluble protein fraction 0.125 with the molecular weight of18-20 kD, isolated from the destruction products of mycobacterium of M.bovis strain No. 8 the phosphate-buffered saline 24.875 the aqueousformaldehyde solution 0.037, the isotonic sodium chloride solution therest.

Similarly, a remedy was prepared from protein fractions with proteincontents of 0.48-0.52%.

Example 2. Substantiation of the Quantitative Content of Components inthe Remedy

120 two to nine days old calves were used in our experiments, of which108 were injected at doses of 4 to 6 ml to prevent leukemia withdifferent quantitative contents of the components.

12 animals formed a reference group, which were injected pure saline ina dose of 5 ml per capita. The results are shown in Table 2.

TABLE 2 Quantitative contents of components, wt % Phosphate- Formal-Isotonic Dose of Protein buffered dehyde NaCl preparation, Number DiedSurvived fraction saline solution solution mL of calves Nos % Nos % 0.059.95 0.037 Rest 4 12 0 0 12 100 to 5 12 1 8.3 11 91.7 100% 6 12 3 25.0 975.0 0.075 14.925 0.037 Rest 4 12 2 16.7 10 83.3 to 5 12 0 0 12 100 100%6 12 2 16.7 10 83.3 0.125 24.875 0.037 Rest 4 12 0 0 12 100 to 5 12 0 012 100 100% 6 12 1 8.3 11 91.7

4 animals of 12 were lost in the reference group, which was 33.3%.

Example 3

To substantiate the increased number of killer cells in the body ofanimals (heifers and cows), immunological studies of the blood ofanimals treated at the age of 2-9 days with our remedy to prevent bovineleukemia were carried out:

2.5 years old heifers, and

5 years old cows.

The results are shown in Table 3.

TABLE 3 Immunological indicators of 2.5 years old heifers and 5 yearsold cows Group T-lymph. Th-lymph. Ts-lymph. Th/Ts T-active. B-lymph. Th02.5 years old HEIFERS Non-  3343 ± 286.07  2054 ± 178.18  1295 ± 113.84 1.6 ± 0.07 1876 ± 159.95   459 ± 38.29  877 ± 97.93 treated Treated 3081 ± 249.9  1522 ± 133.1  1559 ± 132.3  1.0 ± 0.14 1631 ± 133.51  517± 39.5  1616 ± 242.7 5 years old COWS Non- 2421.14 ± 279.34  1489.0 ±157.85 931.29 ± 127.07 1.66 ± 0.11 1315.43 ± 154.77   288.43 ± 32.89744.43 ± 110.62 treated Treated 4028.0 ± 586.89 2369.57 ± 345.5  1660.71± 241.39  1.43 ± 0.04 2167.86 ± 318.2    528.29 ± 68.81 1406.0 ± 203.93

From Table 3 it follows that the 2.5 years old heifers show a decreasedratio of T-helpers to T-suppressors (Th/Ts) and an increased number ofthe third subpopulation of T-lymphocytes (Th0).

In the 5 years old (experimental) cows, a significant increase in thecontents of all lymphocyte fractions (T and B) was recorded whilemaintaining a high content of Th0 (killers).

The results of our serological tests for the presence of antibodies toBLV, carried out as the immunodiffusion reaction, in the heifers andcows aged 2.5 and 5 years, respectively, were negative.

Thus, the claimed remedy for bovine leukemia prevention, as well as themethod of use thereof, ensures lifelong resistance of animals toleukemia infection by activating the cellular immunity against BLV byincreasing the number of killer cells.

What is claimed is:
 1. Remedy for bovine leukemia preventioncharacterized in that it contains: a water-soluble protein fraction witha molecular weight of 18-20 kDa, isolated from the destruction productsof tuberculosis mycobacterium and characterized by the presence of peaksnear a wavelength of 214 nm in the UV region, a phosphate-bufferedsaline, an aqueous formaldehyde solution and an isotonic sodium chloridesolution at the following concentrations of the components, wt %: thewater-soluble protein fraction with the 0.05-0.125; molecular weight of18-20 kD, isolated from the tuberculosis mycobacterium destructionproducts the phosphate-buffered saline  9.95-24.875; the formaldehydeaqueous solution 0.025-0.046;  the isotonic sodium chloride solution therest.


2. Remedy for bovine leukemia prevention characterized in that itcontains: a water-soluble protein fraction with a molecular weight of18-20 kDa, isolated from the destruction products of tuberculosismycobacterium and characterized by the presence of peaks near awavelength of 214 nm in the UV region, a phosphate-buffered saline, anaqueous formaldehyde solution and an isotonic sodium chloride solutionat the following concentrations of the components, wt %: thewater-soluble protein fraction with the 0.125; molecular weight of 18-20kD, isolated from the tuberculosis mycobacterium destruction productsthe phosphate-buffered saline 24.875;  the formaldehyde aqueous solution0.037; the isotonic sodium chloride solution the rest.


3. Method of using the remedy for bovine leukemia prevention, comprisingits intramuscular administration to 1 to 9 day old calves at a dose of4.0-6.0 ml per capita, wherein the remedy contains the water-solubleprotein fraction with the molecular weight of 18-20 kDa isolated fromthe destruction products of tuberculosis mycobacterium and characterizedby the presence of peaks near the wavelength of 214 nm in the UV region,the phosphate-buffered saline, the aqueous formaldehyde solution, andthe isotonic solution of sodium chloride at the following componentconcentrations, wt %: the water-soluble protein fraction with the0.05-0.125; molecular weight of 18-20 kD isolated from the tuberculosismycobacterium destruction products the phosphate-buffered saline 9.95-24.875; the aqueous formaldehyde solution 0.025-0.046;  theisotonic sodium chloride solution the rest.